Process of chromatographic extraction of vitamin d2 and/or pre-vitamin d2



, rRocEss or CHRGMATOGRAPHIC EXTRACTION F VITAMIN D AND/0R PIKE-VITAMIN D Francis Balestic, Versailles, France, ,assignor to Commissariat a IEnergie Atomique No Drawing. Filed Mar. 18, 1963, Ser. No. 266,046 Claims priority, applicatziogizgrance, Mar. 29, 1962, 3,

6 Claims. (Cl. 260-39725) y The present invention relates to a process of extraction,

by chromatography on an activated fiuoridine, of vitamin D and/or pre-vitamin D Vitamin D is generally prepared by theultravioletiirradiation of ergosterol in solution in an organic solvent, which must not contain any trace of oxygen or peroxide; The solvent used-is usually benzene, ethyl ether, ethyl alcohol, pentane, hexane or cyclohexane.

The source of ultraviolet rays is generally constituted 'by a magnesium are, operating at a voltage of about 10,000 v., or a mercury yapour lamp. Preferably, the radiation is filtered in order to improve the yield of vitamin D by utilising only radiation having wavelengths of 275-300 microns. 5 7

Onlypre-vitamin D (precalciferol) is formed, if the temperature is lower than 20 C. Between 20: and 80-C., a mixture of calciferol and pre-calciferol is obtained, the proportion of calciferol increasing with temperature. Around 75 C., calciferol principally forms. The risk of decomposition in practice prohibits operation at a temperature of more than 80' C The vitamin and/or pro-vitamin D thus obtained can'- be separated from the irradiated mixture by various methi ods, such as the'following: 1 'L V r l (l) The ergosterol is precipitated in the first place with digitonin. Then, tachysterol and lumi sterol, namely other irradiation products, are eliminated with citraconic anhydride, which, transforms them into insoluble addi tion productsand calciferol is isolated by fractional crystallisation from acetone.

(2) The mixture of irradiation products is esterified with 3.5dinitrobenzoyl chloride in a mixture of benzene and pyridine. The various dinitrobenzoates of the irscrubbingjby an organic solvent.

1 or weakly polar organic solvent, effecting chromatographic adsorption of the solution by passage through an activated fluoridine and eluting the said vitamins with an organic solventwhich is not less polar than the first-mentioned solvent.

' A preferred method for carrying out the activation of the fiuoridine consists in eliminating a large part at least of the iron which it contains, heating for a substantial time period below 100 C., adjustment of the particle size range at from 100 to 500 microns and at least one The treatment for activation of the fluoridine de scribed above enhances its adsorption capacity and resolu-' tion power and favours the decomposition of undesirable irradiation products'on its surface. 7

"Preferably, the treatment of the fluoridine is carried out so that the activated fiuoridineutilised for the adsorp tion is constituted by particles which have dimensions from 275 to 325 microns. An example of the process of chromatographic extraction of vitaminD and/ or previtamin D according to the invention is given below.

The means for carrying out the example should be considered as forming part of the invention, it being .un-

derstoodthat equivalent means can also be used.

radiation products are then separated by chromatographic absorption in a column of neutral alumina and then recrystallised ,from suitable organic solvents (petroleum, ether, methyl ethyl ketone, benzene).

The vitamin and/ or pro-vitamin D can also be formed by the irradiation of ergosterol by means of ionising radiations, and then extracted by methods which are identical or analogous to those set out above for irradiation with ultraviolet rays.

In a general manner, the method of extraction by chromatographic adsorption can utilise, for example, absorbent substances constituted by or based on magnesia (celite magnesia, for example) activated bentonite (super-filtroc), activated neutral alumina, fluoridine, etc. Such a method, already used for the separation of ultraviolet radiation products containing vitamin D can be used for the case of ionisation radiation. Also, it is applicable to the separation of vitamins A and B contained in fish liver oils.

The present invention has the object of providing a process of extraction, by chromatography on an activated fluoridine, of vitamin D and/ or pro-vitamin D obtained by irradiation.

According to the invention, there is provided a process of chromatographic extraction of vitamin D and/or pre-vitamin D prepared by the ultraviolet irradiation of ergosterol, which comprises forming a solution of the irradiation products associated with the vitamin D and/ or pre-vitamin D and unconverted products in nonpolar EXTRACTION OF VITAMIN D AND/OR PRE-VITAMIN D (a) Preparation 01 activajted flu orz'dine and setting up 10 i the adsorption column A fluoridine wa' slused' of the type known under the trademark Florex XXS 60/100. This trademark is used with a Floridafullers earth that has been subject- Q ed to an extrusion process to increase its adsorptive capacity by 20 to 30% over the untreated material. It is a greyish-white powder with an apparent density of 31-35 lb./cu. ft. and a sp. gr. of 2.2-2.4and isavailable in diiierent mesh sizes. This was heated for three hours in the presence of. concentrated hydrochloric acid in order to eliminate the largest part of the iron which itcontains.

1 It was then subjected to prolonged washing with distilled water, in order to eliminate hydrochloric acid. The

:fiuoridine powder was then heated at 40 C. for 48 hours and then passed successively through two screens having meshes which were respectively 500 microns and 100 microns. The powder was then introduced into a chromatography column (length 20 cm., inner diameter 1.4 cm.) and washed with cc. of ethanol. The washing not only maintains an elevated adsorption capacity, but also causes decomposition in the column of tachysterol. A second washing of the fluoridine with 88 cc. of cyclohexane renders the column capable, under the best possible conditions, of being utilised for adsorption chromatography.

(vb) Preparation 10] an organic solution of irradiated ergosterol Irradiated ergosterol, for example, by means of cobalt 60, was dissolved directly in cyclohexane. cc. of cyclohexane per 20 mg. ergosterol are used.

vitamin D were recovered quantitatively and in the pure state in the first 250 cc. of carbon tetrachloride. The

total operation of the chromatography operations (c) and (d) was about 3 hours.

Patented May 25,1965

.r 3 Other solvents can be used for elutionf he made, for example, of benzene (but-this gives a risk of overlapping of the elution peaks for ergosterol, on the one hand, and for vitamin D and/ or pre-vitamin D on the other hand), and cyclohexane (necessity of using a larger volume of eluant than in the case of carbon tetrachloride; cyclohexane has the advantage, however, of giving a very fine separation of ergosterol and vitamin D The solvent used for dissolvingthe irradiatedergosterol" 7 can also be carbon disulphide or normal hexane, instead of cyclohexane. I l f The method of the invention has the advantage, over the method used previously, of being'mucheasierto use and of giving an-appreciable gain in time, taking into account that fractional precipitations from various solvents,

elimination of the solvent and subsequent chemical treat mentsrare no longer, necessary.

On the other'hand, the process of the invention has.

' Mention may.

non-polar or weakly polar organic solvent, efiecting chromatographic adsorption of the solution 'bypassage through Florida fullers earth and eluting the said vitamins with anorganic solvent which is not less polar than the first-mentioned solvent.

2. A process according to claim 1, in which the irradiation products associated with the vitamins D and pre-vitamins D and unconverted products remain on the Florida fullers earth.

, 3. A process according to claim '1, in which use is made of a' Fiorida fullers earth activa ted byflelimination of a major part of the iron contained therein, prolonged heating at a-temperature of not more than 109C. and washing with an organic solvent. p

4. Aprocessaccording to claim 1, inwhich theFl orida fullers earth has a particle size range of 275 to 325 .microns.

5. A process according to claim 1, in mentioned solvent is cyclohexane.

. 6. A process according to claim 1, in which which the firstthe elution solvent is selected from the group consisting of carbon tetrachloride and cyclohexane.

References Cited by theiEx aminer UNITED STATES PATENTS 2,895,971 7/59 Spiegel'etal.; 260397.25

, LEWIS GOTTS, Primary Examiner. 

1. A PROCESS OF CHROMATOGRAPHIC EXTRACTION OF VITAMIN D2 AND PRE-VITAMIN D2 PREPARED BY THE ULTRAVIOLET IRRADIATION OF ERGOSTEROL, WHICH COMPRISES FORMING A SOLUTION OF THE IRRADIATION PRODUCTS ASSOCIATED WITH THE VITAMIN D2 AND PRE-VITAMIN D2 AND UNCONVERTED PRODUCTS IN NON-POLAR OR WEAKLY POLAR ORGANIC SOLVENT, EFFECTING CHROMATOGRAPHIC ADSORPTION OF THE SOLUTION BY PASSAGE THROUGH FLORIDA FULLER''S EARTH AND ELUTING THE SAID VITAMINS WITH AN ORGANIC SOLVENT WHICH IS NOT LESS POLAR THAN THE FIRST-MENTIONED SOLVENT. 